1. Field of the Invention:
This invention relates to an apparatus and a method for removing a ligand from a ligand carrier in fluids and is particularly directed to the reversible removal of oxygen from oxygen carriers in which oxygen is bound to a cobalt or iron ion.
2. Description of the Prior Art:
Many molecules are bound to carriers when they are transported in a biological system. The properties of hemoglobins, hemerythrins and hemocyanins, the naturally occurring oxygen carriers, have been the subject of numerous studies, as documented in Bonaventura et al, J. Am. Zool., 20, 7 (1980) and 20, 131 (1980). In addition to oxygen, these carrier macromolecules also bind carbon monoxide, nitric oxide, hydroxide, cyanide, azide, fluoride, acetate, and formate, among other ligands. Artificial oxygen carriers and their properties in solution are described by a number of researchers. Traylor et al, "Solvent Effects on Reversible Formation and Oxidative Stability of Heme-Oxygen Complexes", J.A.C.S. 96, 5597 (1974) discloses the effect of solvent polarity on oxygenation of several heme-base complexes prepared by reduction with sodium dithionite or a mixture of Pd black and calcium hydride. Crumbliss et al, "Monomeric Cobalt-Oxygen Complexes", Science, 6, June 1969, Volume 164, pp. 1168-1170, discloses Schiff base complexes of Co(II) which form stable cobalt-oxygen species in solution instead of cobalt-oxygen-cobalt bridged complexes. Crumbless et al, "Monomeric Oxygen Adducts of N,N'-Ethylenebis (acetylacetoniminato) ligand-cobalt(III): Preparation and Properties", J.A.C.S. 92, 55 (1970), discloses a series of monomeric molecular oxygen carriers based on cobalt ligand complexes. Dufour et al, "Reaction of Indoles with Molecular Oxygen Catalyzed by Metalloporphyrins", Journal of Molecular Catalysis, 1, 277 (1980), discloses the catalysis of the oxygenation of simple, alkyl-substituted indoles by Co(II), Co(III), and Mn(III) meso-tetraphenyl-porphines wherein a ternary complex O.sub.2 -CoTPP-indole is formed initially. Brault et al, "Ferrous Porphyrins in Organic Solvents: I. Preparation and Coordinating Properties", Biochemistry, 13, 4591 (1974), discloses the preparation and properties of ferrous deutereporphyrin dimethyl ester and ferrous mesotetraphenylporphine in various organic solvents. Chang et al, "Kinetics of Reversible Oxygenation of Pyrroheme-N-[3-(1-imidazolyl)propyl]amide" discloses studies on the oxygenation of pyrroheme-N-[3-(1-imidazolyl)propyl]amide, i.e., a synthesized section of the myoglobin active site. Castro, "Hexa and Pentacoordinate Iron Poryhyrins", Bioinorganic Chemistry, 4, 45-65 (1974), discloses the direct synthesis of hexa and pentacoordinate iron porphyrins, i.e., the prosthetic groups for the active sites of certain cytochrome and globin heme proteins. Chang et al, "Solution Behavior of a Synthetic Myoglobin Active Site", J.A.C.S., 95, 5810 (1973), discloses studies on a synthesized section of the myoglobin active site and indicates that the oxygen binding reaction does not require the protein. Naturally occurring oxygen carriers have been chemically cross-linked and their properties described. Bonsen et al, U.S. Pat. No. 4,053,590, discloses a polymerized, cross-linked, stromal-free, hemoglobin proposed to be useful as a blood substitute. Morris et al, U.S. Pat. No. 4,061,736, discloses intramolecularly cross-linked, stromal-free hemoglobin. Wong, U.S. Pat. No. 4,064,118, discloses a blood substitute or extender prepared by coupling hemoglobin with a polysaccharide material. Mazur, U.S. Pat. No. 3,925,344, discloses a plasma protein substitute, i.e., an intramolecular, cross-linked hemoglobin composition. However, crosslinked hemoglobin produces macromolecular complexes that retain many of hemoglobin's native properties.
However, little attention has been given to suitable means for reversibly removing the oxygen or other ligand from the carrier once it has become bound thereto. U.S. Pat. No. 4,343,715 describes several methods by which unloading of previously known oxygen carriers can be accomplished. When the oxygen carrier is a porphin-containing compound having a Co.sup.2+ or Fe.sup.2+ ion bound in the porphin ring, a chemical alteration which oxidizes the metal ion to the 3+ state is able to release of the bound oxygen. For example, ferricyanide oxidation of hemoglubin to the ferric state, called in the literature methemoglobin, is a chemical means for unloading the absorbed oxygen. In this and other chemical methods it is necessary to use a regeneration cycle to reactivate the oxygen carrier. With hemoglobin, dithionite can be used to reduce the active sites and render the molecules reactive toward oxygen once again. Such chemical methods are disadvantageous for several reasons. First, they require continuous supplies of both chemical oxidizing solutions and chemical reducing solutions if a continuous oxygen extraction is taking place. Second, direct contact between the chemical and the working fluid which is extracting oxygen is required. This results in contamination of the working fluid by the oxidizing and reducing solutions and generally requires some type of separation before the working fluid can be used again. Third, if oxidizing or reducing solutions are prepared in ready-to-use form, they take up great bulk because of the fluid required and are accordingly not suitable for use in a portable apparatus which is designed for continuous operation over a long period of time.
Hemoglobins derived from fish hemoglobins can be unloaded based on the pH sensitivity of specific fish hemoglobins. Irreversible binding of specific cofactors to normal human blood can also render human hemoglobin pH sensitive so that pH changes can lead to oxygen unloading with this system as well. However, this method has the same disadvantages as chemical oxidation and reduction since solutions of the correct pH must be maintained.
Because of the disadvantages of chemical treatment, other unloading processes have also been disclosed. Simply decreasing the oxygen pressure in the environment of the oxygen carrier has been proposed for oxygen unloading. If desired, unloading can be effected indirectly through a semipermeable membrane. In this case, fluid containing an oxygen carrier to which oxygen is bound is passed on one side of a membrane while a pump on the other side of the membrane is utilized to draw a vacuum and pull the oxygen out of the circulating fluid and across the semipermeable membrane into an oxygen storage chamber. Unfortunately, removal of oxygen from carriers by vacuum suffers from some disadvantages. In laboratory tests, only somewhat more than 50% of the loaded oxygen on insolubilized hemoglobin in polyurethane gels was found to be removed by a vacuum. Furthermore, if the oxygen unloading device is used under water, it becomes increasingly more difficult to draw a vacuum and to support the evacuated chamber against the increase of pressure from the outside environment with increasing depth.
For these and other reasons, alternate methods of removing oxygen and other ligands bound to carriers are still needed.